T lymphocytes divide and differentiate when they receive an appropriate activation signal. This signal can be provided by a clonally-distributed, specific antigen receptor (Ti-T3), or through certain broadly distributed cell membrane structures. Molecular biological and in vitro cell culture techniques have been used to investigate the expression of the genes encoding these activation structures, and to study the relationship between structure and function. We have shown by DNA-mediated gene transfer that the two defined T cell receptor heterodimers assemble in a mutually exclusive manner. Thus, in cells possessing all four chains (alpha, beta, gamma, delta) after transfection, only alpha beta and gamma delta dimers were found associated with CD3 on the T cell membrane. In such cells, the two dimers can compete for limiting CD3, leading to rapid degradation of the non-CD3 associated molecules. Analysis of cells possessing competing functional alpha and beta chains unexpectedly revealed striking asymmetries in the efficiency of expression of certain chain combinations. The variation between two given combinations could be as great as 20x. Much of this difference can be attributed to the germline Vbeta segment, implying that this region interacts with the alpha chain in a structurally constrained manner. This observation suggests that the T cell repertoire may be limited in part by the failure of certain alpha beta combinations to yield enough surface receptor for function. Experiments with a CD3 negative murine hybridoma made CD3 positive by TCR gene transfection raised questions about the currently accepted model of receptor mediated signalling. Thus, this reconstituted cell regained the ability to be activated by anti- Thyl or anti-Ly6 monoclonal antibodies, but its response (measured as IL2 secretion) occurred in the absence of detectable changes in the intracellular (Ca2+) or generation of phosphoinositides. These findings suggest that other second signals may play a critical role in gene activation following receptor occupancy. These studies will enhance our knowledge of which cell membrane molecules are involved in triggering T cells to exert regulatory and effector function, and our understanding of the structural basis for specific T cell responses to antigen.